Label-free capacitive assaying of biomarkers for molecular diagnostics

Abstract

The label-free analysis of biomarkers offers important advantages in developing point-of-care (PoC) biosensors. In contrast to label-based methodologies, such as ELISA, label-free analysis enables direct detection of targets without additional steps and labeled reagents. Nonetheless, label-free approaches require high sensitivity to detect the intrinsic features of a biomarker and low levels of nonspecific signals. Electrochemical capacitance, \(C_{\bar \mu }\), is a feature of electroactive nanoscale films that can be measured using electrochemical impedance spectroscopy. \(C_{\bar \mu }\) is promising as an electrochemical transducing signal for the development of high-sensitivity, reagentless and label-free molecular diagnostic assays. We used a proprietary ferrocene (Fc)-tagged peptide that is able to self-assemble onto gold electrodes (thicknesses <2 nm) to which any biological receptor can be coupled. When coupled with biological receptors (e.g., a monoclonal antibody), \(C_{\bar \mu }\) exhibited by the redox-tagged peptide changes as a function of the target concentration. We provide herein the steps for the qualitative and quantitative detection of dengue non-structural protein 1 (NS1) biomarker. Detection of NS1 can be used to diagnose dengue virus infection, which causes epidemics each year in tropical and subtropical regions of the world. Including the pre-treatment of the electrode surface, the analysis takes ~25 h. This time can be reduced to minutes if the electrode surface is fabricated separately, demonstrating that \(C_{\bar \mu }\) is promising for PoC applications. We hope this protocol will serve as a reference point for researchers and companies that intend to further develop capacitive devices for molecular diagnostic assays.

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Fig. 1: Schematic representation of electrode-electrolyte interfaces.
Fig. 2: Representative electrochemical characterization of the Fc-tagged peptide self-assembled monolayer (SAM).
Fig. 3: Capacitance Nyquist plots of the qualitative NS1 assay obtained after 30 min of incubation of the electrode.
Fig. 4: Electrochemical capacitive results of the quantitative NS1 assay.
Fig. 5: Experimental setup for coating silver wire with silver chloride.
Fig. 6: Experimental setup and representative gold characterization.
Fig. 7

Data availability

Data are available from the authors upon request.

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Acknowledgements

B.L.G. and A.S. acknowledge the support of FAPESP for their scholarship (2018/26273-7 and 2016/17185-1, respectively), and P.R.B. acknowledges the support of FAPESP for the financial support (2017/24839-0 and 2017/02974-3). P.R.B. also acknowledges the individual support by CNPq provided to his head of research activities at São Paulo State University.

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Correspondence to Paulo R. Bueno.

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Key references using this protocol

Cecchetto, J. et. al. Biosens. Bioelectron. 151, 111972 (2020): https://www.sciencedirect.com/science/article/pii/S0956566319310498?via%3Dihub

Piccoli, J. et. al. Anal. Chem. 90, 3005–3008 (2018): https://pubs.acs.org/doi/10.1021/acs.analchem.7b05374

Oliveira, R. M. B., Fernandes, F. C. B. & Bueno, P. R. Electrochim. Acta 306, 175–184 (2019): https://www.sciencedirect.com/science/article/pii/S0013468619304906

Baradoke, A. et. al. Anal. Chem. 92, 3508–3511 (2020): https://pubs.acs.org/doi/10.1021/acs.analchem.9b05633

Ben Aissa, S. et. al. Talanta 195, 525–532 (2019): https://www.sciencedirect.com/science/article/pii/S0039914018311779

Key data used in this protocol

Cecchetto, J. et. al. Biosens. Bioelectron. 151, 111972 (2020): https://www.sciencedirect.com/science/article/pii/S0956566319310498?via%3Dihub

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Garrote, B.L., Santos, A. & Bueno, P.R. Label-free capacitive assaying of biomarkers for molecular diagnostics. Nat Protoc 15, 3879–3893 (2020). https://doi.org/10.1038/s41596-020-0390-9

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